USPTO Patents Application 09648692

PATENT
Dolly, J.O., efa/J

SEQUENCE LISTING

Docket No.: 17311 (BOT)
Serial No.: 09/648,692

<110> Dolly, James Oliver
Li , Yan
Chan, C.K.
Aoki, Kei Roger

<120> Activatable Recombinant Neurotoxins

<130> 17311 (BO)

<140> 09/648,692
<141> 2000-08-25

<150> 60/150,710
<151> 1999-08-25

<160> 29

<170> FastSEQ for Windows Version 4.0

<210> 1
<211> 44
<212> DNA

<213> Artificial Sequence
<220>

<223> PCR primer
<400> 1

gactggtgga cagcaagtcg accggaagct ttacgacgat gacg - 44

<210> 2
<211> 44
<212> DNA

<213> Artificial Sequence
<220>

<223> PCR primer

<210> 3
<211> 30
<212> DNA

<213> Artificial Sequence
<220>

<223> PCR primer
<400> 3

<400> 2

cgtcatcgtc gtaaagcttc cggtcgactt gctgtccacc agtc

44

1

PATENT Docket No.: 1 731 1 (BOT)

Dolly, J. O., et al. Serial No.: 09/648,692

aatagatcta gatcattaac agatttagga 30

<210> 4
<211> 27
<212> DNA

<213> Artificial Sequence
<220>

<223> PCR primer
<400> 4

ttctaaagat ctatacattt gataact 27

<210> 5
<211> 27
<212> DNA

<213> Artificial Sequence
<220>

<223> PCR primer
<400> 5

atgtatagat ctttagaata tcaagta 27

<210> 6
<211> 45
<212> DNA

<213> Artificial Sequence
<220>

<223> PCR primer
<400> 6

atcgataagc ttttatcagt cgacccaaca atccagattt ttaga 45

<210> 7
<211> 65
<212> PRT

<213> Artificial Sequence
<220>

<223> Engineered Intrachain loop region for C. tetani
toxin

<400> 7

Ser Lys Leu lie Gly Leu Cys Lys Lys lie lie Pro Pro Thr Asn lie

15 10 15

Arg Glu Asn Leu Tyr Asn Arg Thr Ala Gly Glu Lys Leu Tyr Asp Asp

20 25 30

Asp Asp Lys Asp Arg Trp Gly Ser Ser Arg Ser Leu Thr Asp Leu Gly

35 40 45

Gly Glu Leu Cys lie Lys Asn Glu Asp Leu Thr Phe lie Ala Glu Lys
50 55 60

Asn

2

PATENT

Dolly, J. O., etal.

Docket No.: 17311 (BOT)
Serial No.: 09/648,692

65

<210> 8
<211> 36
<212> DNA

<213> Artificial Sequence

<220>

<223> PGR primer
<400> 8

aatagaactg caggagaaaa gctttacgac gatgac 36

<210> 9
<211> 36
<212> DNA

<213> Artificial Sequence
<220>

<223> PCR primer

<400> 9

gtcatcgtcg taaagctttt ctcctgcagt tctatt 36

<210> 10
<211> 4017
<212> DNA

<213> Clostridium botulinum

<400> 10

gaattcaagt

tttatctcta

aatcaagatt

ctatggatat

aatagtttta

ggttgtcaag

agaaatgtaa

gatagtagtt

aaaatagtca

gaagaactgt

catattggtg

ttacctaatg

atttctctaa

acattctcac

gatcctgctc

aaagggatta

agaggtacaa

agtgctcagt

aaacttagca

gcaaagtatg

aatgatattt

gttaaatgta

aatgattcta

agaggacaga

agtagataat
actgtttaac
acaattgggt
taaaaaatat
attataatga
aattttataa
ttggtacaac
attatgaccc
caaaaatatt
caaaagctaa
atgcatcagc
ttattataat
gaaataatta
ctgaatattc
ttacattaat
ctacaaagta
atattgaaga
ccaatgatat
aagtacaagt
gattagataa
ttaaaaaatt
ggcaaactta
tttataatat
atgcaaattt

aaaaataatg
tttaacttat
tatatgtgat
ataaatttaa
tcctgttaat
atcatttaat
cccccaagat
taattattta
taatagaata
tccatattta
agttgagatt
gggagcagag
tatgccaagc
ttttagattt
gcatgaatta
tactataaca
attcttaact
ctatactaat
atctaatcca
agatgctagc
atacagcttt
tattggacag
atcagaaggc
aaatcctaga

ccacagattt
aacaatgtaa
cttaatcatg
aattaggaga
gatagaacaa
attatgaaaa
tttcatccgc
caaagtgatg
aataataatc
gggaatgata
aaattctcaa
cctgatttat
aatcaccgtt
aatgataatt
atacattcat
caaaaacaaa
tttggaggta
cttctagctg
ctacttaatc
ggaatttatt
acggaatttg
tataaatact
tataatataa
attattacac

ttattattaa
atatatattt
atataccaaa
tgctgtatat
ttttatatat
atatttggat
ctacttcatt
aagaaaagga
tttcaggagg
atactccaga
atggtagcca
ttgaaactaa
ttggatcaat
gtatgaatga
tacatggact
atcccctaat
ctgatttaaa
attataaaaa
cttataaaga
cggtaaatat
atttacgaac
tcaaactttc
ataatttaaa
caattacagg

taatgatata
gtctataaaa
aaagtcatat
gccaaaaatt
taaaccaggc
aattccagag
aaaaaatgga
tagattttta
gattttatta
taatcaattc
agacatacta
cagttccaat
agctatagta
atttattcaa
atatggggct
aacaaatata
cattattact
aatagcgtct
tgtttttgaa
aaacaaattt
taaatttcaa
aaacttgtta
ggtaaatttt
tagaggacta

60
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480
540
600
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■ 720
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3

PATENT

Dolly, J. O., etal.

Docket No.: 17311 (BOT)
Serial No.: 09/648,692

gtaaaaaaaa tcattagatt ttgtaaaaat attgtttctg taaaaggcat aaggaaatca 1500

atatgtatcg aaataaataa tggtgagtta ttttttgtgg cttccgagaa tagttataat 1560

gatgataata taaatactcc taaagaaatt gacgatacag taacttcaaa taataattat 1620

gaaaatgatt tagatcaggt tattttaaat tttaatagtg aatcagcacc tggactttca 1680

gatgaaaaat taaatttaac tatccaaaat gatgcttata taccaaaata tgattctaat 1740

ggaacaagtg atatagaaca acatgatgtt aatgaactta atgtattttt ctatttagat 1800

gcacagaaag tgcccgaagg tgaaaataat gtcaatctca cctcttcaat tgatacagca 1860

ttattagaac aacctaaaat atatacattt ttttcatcag aatttattaa taatgtcaat 1920

aaacctgtgc aagcagcatt atttgtaagc tggatacaac aagtgttagt agattttact 1980

actgaagcta accaaaaaag tactgttgat aaaattgcag atatttctat agttgttcca 2040

tatataggtc ttgctttaaa tataggaaat gaagcacaaa aaggaaattt taaagatgca 2100

cttgaattat taggagcagg tattttatta gaatttgaac ccgagctttt aattcctaca 2160

attttagtat tcacgataaa atctttttta ggttcatctg ataataaaaa taaagttatt 2220

aaagcaataa ataatgcatt gaaagaaaga gatgaaaaat ggaaagaagt atatagtttt 2280

atagtatcga attggatgac taaaattaat acacaattta ataaaagaaa agaacaaatg 2340

tatcaagctt tacaaaatca agtaaatgca attaaaacaa taatagaatc taagtataat 2400

agttatactt tagaggaaaa aaatgagctt acaaataaat atgatattaa gcaaatagaa 2460

aatgaactta atcaaaaggt ttctatagca atgaataata tagacaggtt cttaactgaa 2520

agttctatat cctatttaat gaaaataata aatgaagtaa aaattaataa attaagagaa 2580

tatgatgaga atgtcaaaac gtatttattg aattatatta tacaacatgg atcaatcttg 2640

ggagagagtc agcaagaact aaattctatg gtaactgata ccctaaataa tagtattcct 2700

tttaagcttt cttcttatac agatgataaa attttaattt catattttaa taaattcttt 2760

aagagaatta aaagtagttc agttttaaat atgagatata aaaatgataa atacgtagat 2820

acttcaggat atgattcaaa tataaatatt aatggagatg tatataaata tccaactaat 2880

aaaaatcaat ttggaatata taatgataaa cttagtgaag ttaatatatc tcaaaatgat 2940

tacattatat atgataataa atataaaaat tttagtatta gtttttgggt aagaattcct 3000

aactatgata ataagatagt aaatgttaat aatgaataca ctataataaa ttgtatgaga 3 060

gataataatt caggatggaa agtatctctt aatcataatg aaataatttg gacattcgaa 3120

gataatcgag gaattaatca aaaattagca tttaactatg gtaacgcaaa tggtatttct 3180

gattatataa ataagtggat ttttgtaact ataactaatg atagattagg agattctaaa 3240

ctttatatta atggaaattt aatagatcaa aaatcaattt taaatttagg taatattcat 3300

gttagtgaca atatattatt taaaatagtt aattgtagtt atacaagata tattggtatt 3360

agatatttta atatttttga taaagaatta gatgaaacag aaattcaaac tttatatagc 3420

aatgaaccta atacaaatat tttgaaggat ttttggggaa attatttgct ttatgacaaa 3480

gaatactatt tattaaatgt gttaaaacca aataacttta ttgataggag aaaagattct 3540

actttaagca ttaataatat aagaagcact attcttttag ctaatagatt atatagtgga 3600

ataaaagtta aaatacaaag agttaataat agtagtacta acgataatct tgttagaaag 3660

aatgatcagg tatatattaa ttttgtagcc agcaaaactc acttatttcc attatatgct 3720

gatacagcta ccacaaataa agagaaaaca ataaaaatat catcatctgg caatagattt 3780

aatcaagtag tagttatgaa ttcagtagga aattgtacaa tgaattttaa aaataataat 3840

ggaaataata ttgggttgtt aggtttcaag gcagatactg tcgttgctag tacttggtat 3900

tatacacata tgagagatca tacaaacagc aatggatgtt tttggaactt tatttctgaa 3960

gaacatggat ggcaagaaaa ataaaaatta gattaaacgg ctaaagtcat aaattcc 4017

<210> 11
<211> 37
<212> DNA
<213> Artifical

Sequence

<220>

<223> PCR primer
<400> 11

cccggatccc caaaaattaa tagttttaat tataatg 37

4

PATENT

Dolly, J. O., et al.

Docket No.: 17311 (BOT)
Serial No.: 09/648,692

<210> 12

<211> 36

<212> DNA

<213> PCR primer

<400> 12

cccctgcagt catttttctt gccatccatg ttcttc 36

<210> 13
<211> 31
<212> DNA

<213> Artificial Sequence
<220>

<223> PCR primer
<400> 13

cagttaatac attcattaca tggactatat g 31

<210> 14
<211> 26
<212> DNA

<213> Artificial Sequence
<220>

<223> PCR primer
<400> 14

atgcattaat gtaagagcag gatctt 26

<210> 15
<211> 5
<212> PRT
<213> Site

<220>

<221> SITE

<222> (1) . . . (5)

<223> protease cleavage site

<400> 15

Asp Asp Asp Asp Lys
1 5

<210> 16
<211> 8
<212> PRT
<213> Site

<220>

<221> SITE

<222> (1) . . . (8)

<223> Protease cleavage site

5

PATENT

Dolly, J. O., etal.

Docket No.: 17311 (BOT)
Serial No.: 09/648,692

<400> 16

Leu Glu Val Leu Phe Gin Gly Pro
1 5

<210> 17
<211> 5
<212> PRT

<213> Clostridium species
<220>

<221> ZN_FING

<222> (1) . . . (5)

<223> Xaa=any amino acid

<400> 17

His Glu Xaa Xaa His
1 5

<210> 18
<211> 51
<212> DNA

<213> Artificial Sequence
<220>

<223> Linker
<400> 18

ggagaaaagc tttacgacga tgacgataag gatcgatggg gatcctctag a 51

<210> 19
<211> 22
<212> PRT

<213> Artificial Sequence
<220>

<223> Linker
<400> 19

Met Gly Gly Ser His His His His His His Gly Met Ala Ser Met Thr

1 5 10 15

Gly Gly Gin Gin Val Asp
20

<210> 20
<211> 19
<212> PRT

<213> Clostridium botulinum
<400> 20

Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys lie Lys lie Lys Asn Glu

15 10 15

Asp Leu Thr

6

PATENT

Dolly, J. O., &t al.

Docket No.: 17311 (BOT)
Serial No.: 09/648,692

<210> 21
<211> 54
<212> DNA

<213> Artificial Sequence
<220>

<223> Linker
<400> 21

atgagaggat cgcatcacca tcaccatcac ggatccccaa aaattaatag tttt 54

<210> 22
<211> 7
<212> PRT

<213> Artificial Sequence
<220>

<221> SITE

<222> (2) ... (3)

<223> Xaa=any amino acid

<223> Protease cleavage site

<400> 22

Glu Xaa Xaa Tyr Ser Gin Ser
1 5

<210> 23
<211> 7
<212> PRT

<213> Artificial Sequence
<220>

<221> SITE

<222> (2) ... (3)

<223> Xaa=any amino acid

<221> SITE

<222> (5) . . . (5)

<223> Xaa=any amino acid

<223> Protease cleavage site

<400> 23

Glu Xaa Xaa Tyr Xaa Gin Gly
1 5

<210> 24
<211> 18
<212> PRT

7

PATENT

Dolly, J. O., etai

Docket No.: 17311 (BOT)
Serial No.: 09/648,692

<213> Artificial Sequence
<220>

<223> Translated PCR fragment
<400> 24

Met Arg Gly Ser His His His His His His Gly Ser Pro Lys lie Asn

15 10 15

Ser Thr

<210> 25
<211> 5
<212> PRT

<213> Clostridium species
<220>

<221> ZN_FING
<222> (1) . . . (5)

<400> 25

His Glu Leu lie His
1 5

<210> 26
<211> 5
<212> PRT

<213> Clostridium botulinum C
<220>

<221> ZN_FING
<222> (1) . . . (5)

<400> 26

His Glu Leu Asn His
1 5

<210> 27
<211> 5
<212> PRT

<213> Clostridium botulinum D
<220>

<221> ZN_FING
<222> (1) . . . (5)

<400> 27

His Glu Leu Thr His
1 5

<210> 28

8

PATENT

Dolly, J. O., etai

Docket No.: 17311 (BOT)
Serial No.: 09/648,692

<211> 4
<212> PRT

<213> Homo sapiens
<220>

<221> SITE
<222> (1) . . . (4)

<223> Blood coagulation factor Xa

<400> 28
lie Glu Gly Arg
1

<210> 29
<211>' 17
<212> PRT

<213> Artifical sequence
<220>

<223> Linker
<400> 29

Gly Glu Lys Leu Tyr Asp Asp Asp Asp Lys Asp Arg Trp Gly Ser Ser

15 10 15

Arg

9